The principal objective of this work is to design isozyme-specific activator/inhibitors of protein kinase C (PK-C) capable of binding selectively to the regulatory, C1 domain of PK-C isozymes and other families of transducing enzymes with structurally similar C1 domains. The main synthetic effort is to design, simple and chemically accessible compounds that are structurally related to the natural PK-C agonist, the second messenger diacylglycerol (DAG). A combinatorial approach that utilizes a 4,4-disubstituted-gamma-butyrolactone template as a conformationally rigid DAG scaffold (DAG-lactones) was implemented to investigate the best combination of alkyl and acyl chains to improve hydrophobic interactions with a group of highly conserved hydrophobic amino acids along the rim of the C1 domain (e.g., the Phe-243, Leu-250, Trp-252 and Leu-254 in PK-Cdelta). The combination of linear and branched chains produced a large library of compounds where optimal ligands reached namolar binding affinities. The best compound yet discovered, (Z)-{1-(Hydroxymethyl)-4-[4-methyl-3- (methylethyl)pentylidene]-3-oxo-2-oxolanyl}methyl 4-methyl-3- (methylethyl)pentanoate, is a doubly branched DAG-lactone with low namomolar (ca. 2 nM) binding affinity for PK-C. This compound showed potent and selective antitumor activity in the NCI 60-cell line in vitro screen against specific breast, colon and leukemia cell lines at low nanomolar concentrations. Besides binding to the C1 domains of the classical and novel members of PK-C isozymes, DAG and the phorbol esters are also able to bind to the C1 domain(s) in other families of proteins involved in signal transduction, namely PKD, the chimaerins and RasGRP. The compound described above is the first compound ever to surpass phorbol ester in potency (subnanomolar Ki) and selectivity (3- fold) for the C1 domain of beta2-chimaerin. Chimaerins do not possess a functional kinase domain but they are GTPase-activating proteins (GAP) for Rac, a small GTP binding protein of the Ras superfamily. The combinatorial chemical approach described here, coupled to intensive molecular modeling and docking studies of the different C1 domains, bodes well for the future design of specific C1-domain-targeted ligands with promising antitumor activity. - molecular models receptors, phorbol Esters, protein kinase C cell signalling, Zinc fingers,